PROJECT SUMMARY Human development begins at fertilization, progresses through a series of cleavage divisions, compaction of the embryo and formation of the blastocyst; then just prior to or during the early stages of gastrulation, a few cells are set aside or allocated to become the germ cells of the next generation and pass the DNA of one generation to the next. Defects in germ cell differentiation are a common cause of human infertility that afflicts 10-15% of couples. However, at least in part due to a lack of models of early human germ cell development, most of the underlying cellular and molecular correlates of infertility remain unknown. The overall goal of this project is to fill this knowledge void, at least in part, by dissecting the intrinsic transcriptional network in early human germ cells. The hypothesis underlying this proposal is that a unique TF network, comprised of human somatic lineage specifiers in cooperation with pluripotency genes, controls the earliest developmental transition in germ cell differentiation from pluripotent stem cells to human primordial germ cells (hPGCs) by repressing somatic gene expression while activating germ cell programs; moreover, we hypothesize that the same network functions to maintain germ cell identity and promote progression to the spermatogonial stage and beyond. Our preliminary studies have uncovered a unique transcriptional network in human germ cells that is regulated by an OCT4-PAX5-PRDM1 circuit. To address our hypothesis, we propose three specific aims to: 1) Map diagnostic genome-wide localization of TFs in bona fide hPGCs. 2) Dissect cooperativity and epistasis of the TF network (OCT4, SOX17, T and PAX5) in hPGCs by gain- and loss-of-function analysis. 3) Induce human germ cells via defined TFs. The study is innovative in terms of the hypothesis, preliminary data and the combination of tools of stem cell biology, differentiation, human genome editing, genome-wide transcriptional, epigenetic analysis, and computational biology, that we use to accomplish our overall goal. This project is significant in that it will increase our knowledge of human germ cell developmental genetics by illuminating the transcriptional network governing acquisition of cell fate from pluripotency to the germ cell lineage, enable establishment of a robust genetic system that may parallel that of Drosophila and the mouse in terms of the ability to examine complex genotypes and phenotypes, and may also contribute substantially to potential novel strategies in clinical applications in diagnosis and development of novel therapeutics for infertility.